Methylation-directed regulatory networks determine enhancing and silencing of mutation disease driver genes and explain inter-patient expression variation.

BACKGROUND: Common diseases manifest differentially between patients, but the genetic origin of this variation remains unclear. To explore possible involvement of gene transcriptional-variation, we produce a DNA methylation-oriented, driver-gene-wide dataset of regulatory elements in human glioblastomas and study their effect on inter-patient gene expression variation. RESULTS: In 175 of 177 analyzed gene regulatory domains, transcriptional enhancers and silencers are intermixed. Under experimental conditions, DNA methylation induces enhancers to alter their enhancing effects or convert into silencers, while silencers are affected inversely. High-resolution mapping of the association between DNA methylation and gene expression in intact genomes reveals methylation-related regulatory units (average size = 915.1 base-pairs). Upon increased methylation of these units, their target-genes either increased or decreased in expression. Gene-enhancing and silencing units constitute cis-regulatory networks of genes. Mathematical modeling of the networks highlights indicative methylation sites, which signified the effect of key regulatory units, and add up to make the overall transcriptional effect of the network. Methylation variation in these sites effectively describe inter-patient expression variation and, compared with DNA sequence-alterations, appears as a major contributor of gene-expression variation among glioblastoma patients. CONCLUSIONS: We describe complex cis-regulatory networks, which determine gene expression by summing the effects of positive and negative transcriptional inputs. In these networks, DNA methylation induces both enhancing and silencing effects, depending on the context. The revealed mechanism sheds light on the regulatory role of DNA methylation, explains inter-individual gene-expression variation, and opens the way for monitoring the driving forces behind deferential courses of cancer and other diseases.

Embryonic Stem Cell (ES)-Specific Enhancers Specify the Expression Potential of ES Genes in Cancer.

Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers (ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer.

Quantitative functional MRI biomarkers improved early detection of colorectal liver metastases.

PURPOSE: To implement and evaluate the performance of a computerized statistical tool designed for robust and quantitative analysis of hemodynamic response imaging (HRI) -derived maps for the early identification of colorectal liver metastases (CRLM). MATERIALS AND METHODS: CRLM-bearing mice were scanned during the early stage of tumor growth and subsequently during the advanced-stage. Three experienced radiologists marked various suspected-foci on the early stage anatomical images and classified each as either highly certain or as suspected tumors. The statistical model construction was based on HRI maps (functional-MRI combined with hypercapnia and hyperoxia) using a supervised learning paradigm which was further trained either with the advanced-stage sets (late training; LT) or with the early stage sets (early training; ET). For each group of foci, the classifier results were compared with the ground-truth. RESULTS: The ET-based classification significantly improved the manual classification of the highly certain foci (P < 0.05) and was superior compared with the LT-based classification (P < 0.05). Additionally, the ET-based classification, offered high sensitivity (57-63%), accompanied with high positive predictive value (>94%) and high specificity (>98%) for suspected-foci. CONCLUSION: The ET-based classifier can strengthen the radiologist's classification of highly certain foci. Additionally, it can aid in classifying suspected-foci, thus enabling earlier intervention which can often be lifesaving.

Hemodynamic response magnetic resonance imaging: application for renal hemodynamic characterization.

BACKGROUND: The clinical use of iodinated radiocontrast agents or gadolinium for renal perfusion imaging is limited in the presence of renal dysfunction. We have previously demonstrated the feasibility of hemodynamic response imaging (HRI), a functional magnetic resonance imaging (MRI) method combined with hypercapnia and hypercapnic-hyperoxia, for monitoring changes in liver perfusion and hemodynamics. The aim of the present study was to evaluate the utility of HRI for monitoring changes in renal perfusion and hemodynamics. METHODS: Renal HRI maps were acquired during graded hypercapnia (95% air + 5% CO2) and hypercapnic-hyperoxia (95% O2 + 5% CO2) in control mice. The utility of HRI for monitoring changes in renal perfusion and oxygenation was evaluated using pharmacological inhibition of nitric oxide synthase and cycloxygenase as well as in rhabdomyolysis-induced acute kidney injury (AKI) in mice. HRI results were further interpreted using Doppler ultrasound (US). RESULTS: Renal HRI maps revealed pronounced signal-intensity changes in response to both hypercapnia and hypercapnic-hyperoxia, reflecting intense vascular reactivity. These changes were significantly attenuated following the pharmacological intervention and during AKI, corresponding with hampered perfusion dynamics, as confirmed by Doppler US. CONCLUSIONS: The applicability of the non-invasive HRI method suggests its potential use for the evaluation of renal perfusion and vascular reactivity, excluding the need for contrast-agent administration.

Hemodynamic response imaging: a potential tool for the assessment of angiogenesis in brain tumors.

Blood oxygenation level dependence (BOLD) imaging under either hypercapnia or hyperoxia has been used to study neuronal activation and for assessment of various brain pathologies. We evaluated the benefit of a combined protocol of BOLD imaging during both hyperoxic and hypercapnic challenges (termed hemodynamic response imaging (HRI)). Nineteen healthy controls and seven patients with primary brain tumors were included: six with glioblastoma (two newly diagnosed and four with recurrent tumors) and one with atypical-meningioma. Maps of percent signal intensity changes (ΔS) during hyperoxia (carbogen; 95%O2+5%CO2) and hypercapnia (95%air+5%CO2) challenges and vascular reactivity mismatch maps (VRM; voxels that responded to carbogen with reduced/absent response to CO2) were calculated. VRM values were measured in white matter (WM) and gray matter (GM) areas of healthy subjects and used as threshold values in patients. Significantly higher response to carbogen was detected in healthy subjects, compared to hypercapnia, with a GM/WM ratio of 3.8 during both challenges. In patients with newly diagnosed/treatment-naive tumors (n = 3), increased response to carbogen was detected with substantially increased VRM response (compared to threshold values) within and around the tumors. In patients with recurrent tumors, reduced/absent response during both challenges was demonstrated. An additional finding in 2 of 4 patients with recurrent glioblastoma was a negative response during carbogen, distant from tumor location, which may indicate steal effect. In conclusion, the HRI method enables the assessment of blood vessel functionality and reactivity. Reference values from healthy subjects are presented and preliminary results demonstrate the potential of this method to complement perfusion imaging for the detection and follow up of angiogenesis in patients with brain tumors.

fMRI-based hierarchical SVM model for the classification and grading of liver fibrosis.

We present a novel method for the automatic classification and grading of liver fibrosis based on hepatic hemodynamic changes measured noninvasively from functional MRI (fMRI) scans combined with hypercapnia and hyperoxia. The supervised learning method automatically creates a classification and grading model for liver fibrosis grade from training datasets. It constructs a statistical model of liver fibrosis by evaluating the signal intensity time course and local variance in T2(*)-W fMRI scans acquired during the breathing of air, air-carbon dioxide, and carbogen with a hierarchical multiclass binary-based support vector machine (SVM) classifier. Two experimental studies on 162 slices from 34 mice with the hierarchical multiclass binary-based SVM classifier yield 96.9% separation accuracy between healthy and histological-based fibrosis graded subjects, and an overall accuracy of 75.3% for healthy, fibrotic, and cirrhotic subjects. These results outperform existing image-based methods that can discriminate between healthy and mild-grade fibrosis subjects.

Vascular profile characterization of liver tumors by magnetic resonance imaging using hemodynamic response imaging in mice.

Recently, we have demonstrated the feasibility of using hemodynamic response imaging (HRI), a functional magnetic resonance imaging (MRI) method combined with hypercapnia and hyperoxia, for monitoring vascular changes during liver pathologies without the need of contrast material. In this study, we evaluated HRI ability to assess changes in liver tumor vasculature during tumor establishment, progression, and antiangiogenic therapy. Colorectal adenocarcinoma cells were injected intrasplenically to model colorectal liver metastasis (CRLM) and the Mdr2 knockout mice were used to model primary hepatic tumors. Hepatic perfusion parameters were evaluated using the HRI protocol and were compared with contrast-enhanced (CE) MRI. The hypovascularity and the increased arterial blood supply in well-defined CRLM were demonstrated by HRI. In CRLM-bearing mice, the entire liver perfusion was attenuated as the HRI maps were significantly reduced by 35%. This study demonstrates that the HRI method showed enhanced sensitivity for small CRLM (1-2 mm) detection compared with CE-MRI (82% versus 38%, respectively). In addition, HRI could demonstrate the vasculature alteration during CRLM progression (arborized vessels), which was further confirmed by histology. Moreover, HRI revealed the vascular changes induced by rapamycin treatment. Finally, HRI facilitates primary hepatic tumor characterization with good correlation to the pathologic differentiation. The HRI method is highly sensitive to subtle hemodynamic changes induced by CRLM and, hence, can function as an imaging tool for understanding the hemodynamic changes occurring during CRLM establishment, progression, and antiangiogenic treatment. In addition, this method facilitated the differentiation between different types of hepatic lesions based on their vascular profile noninvasively.

Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is considered to be the outcome of chronic liver inflammation. Currently, the main treatment for HCC is surgical resection. However, survival rates are suboptimal partially because of tumor recurrence in the remaining liver. Our aim was to understand the molecular mechanisms linking liver regeneration under chronic inflammation to hepatic tumorigenesis. Mdr2-KO mice, a model of inflammation-associated cancer, underwent partial hepatectomy (PHx), which led to enhanced hepatocarcinogenesis. Moreover, liver regeneration in these mice was severely attenuated. We demonstrate the activation of the DNA damage-response machinery and increased genomic instability during early liver inflammatory stages resulting in hepatocyte apoptosis, cell-cycle arrest, and senescence and suggest their involvement in tumor growth acceleration subsequent to PHx. We propose that under the regenerative proliferative stress induced by liver resection, the genomic unstable hepatocytes generated during chronic inflammation escape senescence and apoptosis and reenter the cell cycle, triggering the enhanced tumorigenesis. Thus, we clarify the immediate and long-term contributions of the DNA damage response to HCC development and recurrence.

Functional magnetic resonance imaging monitoring of pathological changes in rodent livers during hyperoxia and hypercapnia.

UNLABELLED: Liver diseases and regeneration are associated with hemodynamic changes denoting pathological alterations. Determining and monitoring physiological and pathological liver changes is essential for diagnostic and therapeutic objectives. Our aim was to determine the feasibility of functional magnetic resonance imaging (fMRI) during hypercapnia and hyperoxia for monitoring liver pathology. Liver fMRI images were acquired in rodents following acute bleeding, partial hepatectomy, and fibrosis. Results were quantitated and confirmed by histology. Changes induced by hyperoxia and hypercapnia following hemorrhage significantly correlated with the percentage of blood loss, reflecting lower liver perfusion and diminished vessel responsiveness to gas saturation. Hepatectomy resulted in an early decline in signal intensity changes due to hyperoxia, suggesting a decrease in liver perfusion and blood content. Following hepatectomy, signal intensity changes due to hypercapnia increased, signifying a change in liver perfusion from a mainly portal to a more arterial source. Two weeks after induction of fibrosis, signal intensity changes due to hypercapnia became much lower and those due to hyperoxia were much higher than those in normal livers, reflecting the increased perfusion due to the inflammatory process as confirmed by histologic analysis. With fibrosis progression, signal intensity changes induced by hypercapnia and hyperoxia were gradually attenuated, indicating structural and functional alterations of the liver vasculature during fibrosis. CONCLUSION: In various liver pathologies, fMRI response to hypercapnia and hyperoxia is sensitive to changes in liver hemodynamic status involved in hepatic damage or recovery; thus, this technique may offer an additional noninvasive diagnostic tool for evaluation and follow-up of liver diseases by means of examining perfusion-related alterations.

Functional MR imaging during hypercapnia and hyperoxia: noninvasive tool for monitoring changes in liver perfusion and hemodynamics in a rat model.

PURPOSE: To prospectively assess functional magnetic resonance (MR) imaging during hypercapnia and hyperoxia for monitoring changes in liver perfusion and hemodynamics in rats. MATERIALS AND METHODS: All experiments were performed with approval of an animal care and use committee. Functional T2*-weighted gradient-echo MR images of the rat liver were acquired during hyperoxia and graded hypercapnia (n=24). Additional images were acquired during portal vein ligation (n=4), induced hypovolemia (n=5), and 70% hepatectomy (n=5). Hypercapnic effects were confirmed with Doppler ultrasonography and with gadopentetate dimeglumine. Differences between groups were analyzed by using Wilcoxon rank sum test, except for the graded hypercapnia, for which one-way analysis of variance was used. RESULTS: Liver signal intensity (SI) increased due to hyperoxia; the percentage change in SI was seven times greater than that in muscle tissue; this reflects higher vascularity of the liver. Liver SI decreased due to hypercapnia; the percentage change in SI was negative in the liver but positive in the muscle (P<.001). Induced hypovolemia resulted in considerable decreases in functional MR imaging response; this reflects lower liver perfusion. Clinical applicability of the functional MR imaging method was proved by monitoring changes in liver perfusion that resulted from liver resection. CONCLUSION: In the liver, the magnitude of the percentage change in SI induced by hypercapnia and hyperoxia reflects changes in total blood volume; whereas percentage change in SI values induced by hypercapnia from a negative to a positive value reflects relative changes in portal-to-arterial blood flow ratio.